Development of the olfactory epithelium of mice and cell differentiation

The OE can be classified as a pseudostratified neuroepithelium, that composes various cell types (Treloar et al. 2010) and is attached to a basal lamina. Two subpopulations of basal cells can be distinguished, namely the self-renewing globose basal cells (GBCs) which produce olfactory sensory neurons and non-neuronal cells like sustentacular cells (SUS) (Treloar et al. 2010). Further the horizontal basal cells (HBCs), which are as well multipotent cells, which pos-sess the capability of producing GBCs and thus constitute a supply of long-lived progenitors (Leung et al. 2007; Joiner et al. 2015).

At E10.5 - 11.5, “early neurogenesis” takes place in the OE, in which the first cohort of neurons are generated and migrate out of the OE (Beites et al. 2005).

Up to day E11.5 the largest proportion of the olfactory sensory neurons are sit-uated in a proliferative stadium (Ikeda et al. 2007; Bachmann et al. 2016).

At E12.5, the OE is composed of a pseudostratified epithelium and reveals “es-tablished neurogenesis” (Figure 3) (Beites et al. 2005; Ikeda et al. 2007).

By E13.5 the OE is organized into three divisions (apical, middle and basal), and multiplying cells have established in the apical and basal aspects (Cau et al. 2002; Ikeda et al. 2007). In the basal area, the major amount of cells are stem cells and intermediate progenitors, as that precursors of olfactory receptor neurons (Menini 2010; Gokoffski et al. 2011; Kam et al. 2014; Suzuki and Osumi 2015; Bachmann et al. 2016).

Figure 2: Schematic development of the olfactory epithelium

At E9.5 the olfactory placode starts to thicken; by E10.5 it starts to invaginate to form the olfac-tory pit, the onset of the nasal cavity. By E11.5 the olfacolfac-tory pit has developed to a nasal cavity.

At around E12.5 the OE reveals a pseudostratified epithelium. Until E14 the nasal cavity turns out to be more extensive. Source: Kawauchi et al 2005; with friendly permission of Dr.

Anne Calof

They are able to renew, or to develop either to an intermediate progenitor, a sustentacular cell (Figure 3A), or to an olfactory ensheating cell (OEC) that sur-rounds the olfactory nerve (Beites et al. 2005). The majority of apical multiplying cells are glial cells which are self-regenerative (Beites et al. 2005; Ikeda et al.

2007; Gokoffski et al. 2011; Inagi et al. 2015; Bachmann et al. 2016). The sus-tentacular cells exhibit a glial-like character and occupy similar functions (Farbman 1992; Ikeda et al. 2007).

Accordingly, they act in a neuroprotective manner by expressing detoxification enzymes (Ding and Coon 1988), provide structural support for ORNs (Nomura et al. 2004) and electrically segregate the cells from each other (Farbman 1992). SUS cells permeate the whole extent of the OE.

During development, the olfactory receptor neurons (ORNs) have differentiated from basal progenitors and are located in the intermediate cell compartment in between the basal and apical layer (Beites et al. 2005). Mature ORNs possess one dendrite, which is directed towards the apical side of the OE with about 12 adjacent cilia (Schwob 2002; Ikeda et al. 2007), to receive the sensory input (Figure 3).

Getting sensory input by odorants, the axonal branches of mature ORNs project directly towards the OB via the olfactory nerve layer (ONL) (Komiyama and Luo 2006; Suzuki and Osumi 2015; Bachmann et al. 2016; Huilgol and Tole 2016).

The axonal branches are escorted by a heterogeneous population of migratory cells, which are olfactory marker protein (OMP)- positive cells. Mature ORNs are exceptionally, due to the fact, that they are lifelong renewed during physio-logical turnover (Graziadei and Graziadei 1979; Schwob 2002; Suzuki and Osumi 2015). Thus, the olfactory epithelium of mice serves as a useful role model to reconstruct how neurogenesis is governed at cellular and molecular levels (Kawauchi 2005) and by which means neurons attain different destinies and regulate their correspondence amongst objectives (Kam et al. 2014; Suzuki and Osumi 2015). Due to the good bioaccessibility to ORNs and their already mentioned lifelong self-renewing potency, they constitute objects of interest for accelerated investigations with a potential of therapeutic benefit (Schwob et al.

2017).

Many transcriptional factors (TFs) playing an important role in embryonic and adult OE neurogenesis have already been identified in earlier studies (Beites et al. 2005; Treloar et al. 2010; Suzuki and Osumi 2015; Bachmann et al. 2016).

A particular cluster of these transcription factors guides the cell differentiation and specification from olfactory neuronal stem cells.

Numerous studies in vitro and in vivo have depicted exactly four levels of devel-opment in the neuronal pathway of the OE (Kawauchi 2005).

In the first developmental step, neural stem cells exhibit the transcription factors Sox2, Pax6 and Nestin (Figure 3A) (Donner et al. 2007). The induction of the olfactory placode is controlled by Pax6, Sox2 and Oct-1 (Collinson et al. 2003;

Donner et al. 2007; Kam et al. 2014). Subsequently, the Mash1 and Ngn1 ex-pression is connected with neural progenitors (Cau et al. 2002) and immature olfactory receptor neurons are expressing Tuj, HuCD, GAP43 as well as Lhx2, NCAM and Ctip2 (Arlotta et al. 2008; Enomoto et al. 2011). Finally, mature ORNs are Ctip2, OMP and NCAM positive (Figure 3A). Migratory cells from the olfactory placode which establish the olfactory mass in cooperation with ORNs axons are marked by the gonadotropin-releasing hormone (GnRH), olfactory marker protein (Treloar et al. 2010) and acetylcholine esterase (Suzuki and Osumi 2015).

BAF complexes have been identified to regulate the expression of the above mentioned transcription factors to control gene expression in a cell lineage de-pendent manner (Ronan et al. 2013). However, it is still unsolved and in the fo-cus of my investigations how these TFs interact and, together with chromatin remodeling factors, coordinate OE neurogenesis (Bachmann et al. 2016).The differentiation of stem cells from pluripotent to more distinct stages of develop-ment coheres with epigenetic changes at the level of chromatin structures. The function of the BAF (mSWI/SNF) chromatin remodeling complexes will be illus-trated in the following section.

Figure 3: Schematic depiction of oNSC development and organiza-tion of the pseudostratified olfacto-ry epithelium and axonal projec-tions in wild-type animals

A The different cell types residing in the olfactory epithelium and the corre-sponding marker proteins.

B At early neurogenesis the major number of cells are stem cells, ac-companied by few intermediate pro-genitor cells and immature neurons.

During early neurogenesis there are no SUS to be found.

C In the middle stage from E12.5 – E15.5 cells are arranged in a pseudo-stratified manner.

D In late stages of olfactory system development, axonal projections be-tween OE and OB, respectively olfac-tory cortex have been formed.